U.S. patent number 5,717,778 [Application Number 08/465,089] was granted by the patent office on 1998-02-10 for optical specimen analysis system and method.
Invention is credited to Albert E. Chu, Lian Tao.
United States Patent |
5,717,778 |
Chu , et al. |
February 10, 1998 |
Optical specimen analysis system and method
Abstract
A method and system for automatically analyzing the presence of
an analyte in a liquid specimen of biological origin are described.
The liquid specimen is added to a testing substrate that has a
receptor immobilized on a portion of its surface. If analyte is
present in the specimen, it will specifically bind to the receptor.
A labeled reagent is added to the testing substrate which will bind
to the analyte, if present, and generate a color on the testing
substrate. The testing substrate is then illuminated and, using
electronic equipment, a digital image of its surface is acquired
and automatically scanned to locate an area that has the highest
color density, which corresponds to the presence of labeled
reagent. A measurement of color density, corresponding to pixels
per unit area, is generated. An area peripheral to the area of
highest color density, which represents background density, is also
located and a measured. The presence or absence of analyte in the
liquid sample is determined by adjusting the measurement of labeled
reagent with the background density measurement in accordance with
a predefined mathematical function. In some cases, the result of
the test will be interpreted as a binary, positive/negative result
according to whether the measurement taken is above or below a
given threshold. In other cases, a continuous range of values may
be generated from different samples that correspond to the
concentration of the analyte present in each tested sample.
Inventors: |
Chu; Albert E. (Hillsborough,
CA), Tao; Lian (San Francisco, CA) |
Family
ID: |
21813206 |
Appl.
No.: |
08/465,089 |
Filed: |
June 5, 1995 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
|
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23113 |
Feb 26, 1993 |
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Current U.S.
Class: |
382/133;
382/274 |
Current CPC
Class: |
G01N
21/8483 (20130101); G06T 7/90 (20170101); G01N
15/1468 (20130101); G01N 15/1475 (20130101); G01N
2021/177 (20130101); G01N 2021/5919 (20130101); G01N
2021/593 (20130101); G01N 2021/5934 (20130101); G01N
2021/8488 (20130101); G06T 2207/10024 (20130101); G06T
2207/30004 (20130101); G01N 2021/5923 (20130101) |
Current International
Class: |
G01N
21/86 (20060101); G06T 7/40 (20060101); G01N
15/14 (20060101); G01N 21/59 (20060101); G06K
009/00 () |
Field of
Search: |
;382/110,128,129,130,133,134,162,164,165,173,175,180,181,190,224,274,287,300,321
;436/43,538,164,805,807,544,536 ;422/67,73,82.05,82.08,82.09,56,57
;352/213 ;356/23,246,39 ;364/413.13 |
References Cited
[Referenced By]
U.S. Patent Documents
Other References
McGraw-Hill Encyclopedia of Science and Technology (13), 7th
Edition, Published by McGraw-Hill, Inc., New York, (1997), pp.
411-421 (No Author). .
McGraw-Hill Yearbook of Science and Technology (1997), Published by
McGraw-Hill, New York. (No Author) (No Page Number)..
|
Primary Examiner: Boudreau; Leo
Assistant Examiner: Tadayon; Bijan
Attorney, Agent or Firm: Flehr Hohbach Test Albritton &
Herbert, LLP
Parent Case Text
REFERENCE TO RELATED APPLICATIONS
This is a continuation-in-part application of U.S. Ser. No.
08/023,113 filed Feb. 26, 1993, now abandoned.
Claims
What is claimed is:
1. A method for determining the presence of at least one biological
analyte that may be present in a liquid sample comprising:
a) applying said liquid sample to a testing substrate having at
least one receptor immobilized thereon at a first receptor area of
said testing substrate, said at least one receptor being capable of
specifically binding directly or indirectly to said at least one
biological analyte, said first receptor area being located in a
limited region of said testing substrate;
b) applying to said testing substrate a reagent that is capable of
specifically binding directly or indirectly to said at least one
biological analyte and capable of generating a color at said first
receptor area when said at least one biological analyte is
present;
c) illuminating said testing substrate and acquiring a digital
image of said testing substrate;
d) using data processing equipment,
(i) automatically scanning said digital image to locate an
optically densest portion of said first receptor area and
generating a first measurement of the density of said densest
portion;
(ii) locating an area peripheral to said first receptor area and
generating a second measurement of background density of said area
peripheral to said first receptor area; and
(iii) generating an output signal by adjusting said first
measurement with said second measurement in accordance with a
predefined mathematical function in order to generate an output
signal that indicates whether said at least one biological analyte
is present in said liquid sample.
2. The method of claim 1 wherein said method is also used for the
determination of a second biological analyte, said testing
substrate having a second receptor immobilized thereon at a second
receptor area, said second receptor area being capable of
specifically binding directly or indirectly to said second
biological analyte, said step (d) including locating and measuring
the density of said first receptor area and said second receptor
area and generating an output signal for said first receptor area
and said second receptor area.
3. The method of claim 1 wherein the output signal of step (d)(iii)
corresponds to the concentration of said at least one biological
analyte present in said liquid sample.
4. The method of claim 1 wherein said testing substrate is a porous
membrane.
5. The method of claim 1, wherein said testing substrate is opaque
and includes a marker having a predefined spatial relationship to
said first receptor area; said step (d)(i) including locating said
marker in said digital image and then locating said first receptor
area in said digital image based on said marker's location.
6. The method of claim 1, further including:
prior to step (d), measuring the optical density of a reference
substrate and calibrating all subsequent measurements of testing
substrates in accordance with said reference substrate's measured
optical density.
7. The method of claim 1, wherein said digital image comprises an
N.times.M array of pixels; said step (d)(i) including:
(1) for each of a selected set of pixel positions spaced apart from
each other, measuring the optical density of a region of said
digital image associated with said each pixel position; (2)
selecting a plurality of said regions measured in step (d)(i)(1)
with highest density, (3) interpolating the pixel positions
associated with said plurality of regions selected in step
(d)(i)(2) to generate a final pixel position, and (4) measuring the
optical density of a region of said digital image associated with
said final pixel position to generate said first measurement.
8. A method for determining the presence of an analyte that may be
present in a liquid sample comprising:
a) applying said liquid sample to a testing substrate having a
receptor immobilized thereon at a receptor area of said testing
substrate, said receptor capable of directly or indirectly binding
to said analyte, said receptor area being located in a limited
region of said testing substrate;
b) applying to said testing substrate a reagent capable of binding
directly or indirectly to said analyte and capable of generating a
color at said receptor area when said analyte is present;
c) illuminating said testing substrate and acquiring a digital
image of said testing substrate;
d) using data processing equipment,
(i) automatically scanning said digital image to locate a portion
of said receptor area having greatest density of a predefined
color, and generating a first measurement of the density of said
predefined color at said located portion;
(ii) locating an area peripheral to said receptor area and
generating a second measurement of background density of said
predefined color at said area peripheral to said receptor area;
and
(iii) generating an output signal by adjusting said first
measurement with said second measurement in accordance with a
predefined mathematical function in order to generate an output
signal that indicates whether said analyte is present in said
liquid sample.
9. The method of claim 8 wherein said method is for the
determination of more than one analyte, said testing substrate
having immobilized thereon more than one receptor area wherein each
receptor area is capable of binding directly or indirectly to a
specific analyte, said step (d) including locating and measuring
the density of said predefined color at each receptor area, and
generating an output signal for each receptor area.
10. The method of claim 8 wherein the adjusted measurement of step
(d)(iii) corresponds to the concentration of analyte present in
said sample.
11. The method of claim 8 wherein said testing substrate is a
porous membrane.
12. A system for analyzing at least one analyte derived from a
biological specimen, wherein said at least one analyte is bound to
a first distinct receptor area of testing substrate wherein a
portion of said testing substrate is not covered by said first
distinct receptor area, said system comprising:
means for acquiring a digital image of said testing substrate;
and
data processing means, coupled to said image acquiring means, for
automatically scanning said digital image to locate an optically
densest portion of said digital image depicting said at least one
analyte and to locate a background portion of said digital image
not depicting said at least one analyte;
said data processing means including means for (A) generating a
first measurement of the density of said densest portion, (B)
generating a second measurement of the density of said background
portion of said testing substrate, and (C) generating an output
signal by adjusting said first measurement with said second
measurement in accordance with a predefined mathematical
function.
13. The system, as defined in claim 12, further including:
means of illuminating said testing substrate; and
means for sensing the intensity of said means for illumination;
said data processing means including means, coupled to said
intensity sensing means, for calibrating said measurements in
accordance with said sensed intensity of said means for
illumination.
14. The system, as defined in claim 12, wherein:
a multiplicity of distinct analytes are bound to a multiplicity of
distinct receptor areas of said testing substrate;
said data processing means including means for locating an
optically densest portion of each said distinct analyte, and for
generating a first measurement of density of said optically densest
portion of each said distinct analyte.
15. The system, as defined in claim 12, wherein said testing
substrate includes a marker having a predefined spatial
relationship to said at least one analyte; and
said data processing means includes means for locating said marker
in said digital image and for locating said at least one analyte
within said digital image based on said marker's location.
16. The system, as defined in claim 12, further including:
a template interposed between said testing substrate and said means
for acquiring a digital image, said template forming a superimposed
image on said digitized image of said testing substrate;
a plurality of guide rails sized to receive said testing substrate
so that said image of said template falls at predefined position
with respect to said testing substrate, thereby indicating where
said analyte is deposited on said testing substrate.
Description
FIELD OF INVENTION
The present invention relates generally to systems and methods of
analyzing specimens, preferably specimens of biological origin, and
particularly to computerized methods and systems for optical
analysis of such specimens.
BACKGROUND OF THE INVENTION
Many laboratory tests are determined by how a particular analyte
present in a sample reacts with a specific reagent. Often, these
tests are qualitatively determined by visual inspection. For
example, current home pregnancy tests can detect human chorionic
gonadotropin (HCG), a hormone secreted in the urine of pregnant
women. Typically, the urine is applied to a testing substrate that
has an antibody immobilized thereon that is capable of binding to
the HCG. A labelled reagent is then applied to the testing
substrate that is capable of specifically binding to HCG, and thus,
in the presence of HCG, will color the testing substrate, thus
indicating a potential pregnancy. For such tests, the user can tell
the results at a glance. However, with increasing demand for rapid
diagnostic testing in a laboratory setting, visual inspection by a
human technician becomes a bottleneck. In addition, human
technicians are error prone, especially when performing diagnostic
tests that require quantitative measurements of color or optical
density. Furthermore, quantifying test results is very difficult
for human technicians when the color density of the testing
substrate varies due to differences in the amount of reagent used
for each test, the amount of liquid (such as blood plasma)
deposited on the substrate, or by different batches of reagents
used in the test. Additionally, the results of such tests often
depend on the difference in color saturation associated with a
specific region of the testing substrate where the analyte
specifically binds compared to another region of the testing
substrate where the analyte is not supposed to bind and thus where
the presence of color indicates the degree of "background" or
"noise" in the test. This is particularly true for rapid
immunoassays that use membranes as the testing substrates that have
discrete zones having receptors immobilized thereon that
specifically bind to the analyte present in the sample tested.
Consequently, a number of prior art systems have been designed to
partially automate the process of quantifying test results. For
example, the results of electrophoretic immunoblots (Western
Blots), the main method for verification of human immunodeficiency
virus seropositivity, can be quantitated using densitometry.
Typically with a Western Blot, bands of electrophoresed proteins
are transferred to a nitrocellulose strip and then incubated with
patient sera. If antibodies specific to the proteins are present in
the sera, they will bind to the blotted proteins. The presence of
the antibodies can be detected using labelled antibodies to human
IgG. If labels are used that generate a visible color, bands will
appear that correspond to the location of the blotted proteins for
which the patient has antibodies. The concentration of the
antibodies can be quantitated using a densitometer, an instrument
which measures optical density by measuring the intensity of
reflected light. The nitrocellulose strip is passed through a beam
of light, so that the intensity of each band is measured and a
value is generated that correlates to the concentration of antibody
present in the patient sera. However, the densitometer only
measures one point of each band as opposed to scanning the entire
area of the band. Because the color intensity of a band can vary,
as well as the background color surrounding each band, the values
generated by densitometry can vary and thus may not accurately
reflect the true concentration of the substance being measured.
Reflectometers are also used to quantitate the results of certain
laboratory tests, particularly, rapid immunoassays such as those
described in U.S. Pat. Nos. 5,006,464 to Chu et al. and 4,632,901
to Valkirs et al., which, after the performance of assay steps, can
result in the appearance of a colored region on a testing substrate
to indicate the presence of a particular analyte in a sample. The
reflectometer is a photoelectric instrument for measuring the
optical reflectance of a surface. Typically, the rapid immunoassay
comprises a testing substrate such as a porous membrane. A small
portion of the testing substrate has a receptor immobilized thereon
(i.e. the receptor area--usually a small circular area or dot) that
is capable of binding directly or indirectly to an analyte such as
an antibody, protein, hormone, or any other substance that is
suspected of being present in a patient sample. Thus, when a
patient sample, such as plasma or urine, comes in contact with the
testing substrate, the analyte, if present in the sample, will bind
specifically to the receptor area of the testing substrate, but not
to the peripheral area of the testing substrate where no receptor
is immobilized. The remainder of the sample and any unbound analyte
will flow through the testing substrate, if it is porous, and/or
can be washed off. A labeled reagent is added that is capable of
binding directly or indirectly to the analyte to generate a colored
dot or circle (or whatever shape the receptor area is). Thus, if
the analyte is present in the patient sample, it will bind to the
receptor area and its presence will be indicated by the generation
of color after application of the labeled reagent.
The results of the immunoassay can then be measured using a
reflectometer. Typically, the testing substrate needs to be
inserted into the reflectometer so that the receptor area will
align with a beam of light that is used to measure reflectance.
Therefore, if the receptor area is not accurately positioned on the
testing substrate, the results of the assay will not be accurately
measured. Additionally, there may be variation in the color
intensity generated at the receptor area. Thus, the beam of light
may not line up with the part of the receptor area that most
accurately correlates to the concentration of analyte present in
the patient sample.
Digital analysis has been used in some testing procedures, but has
not been used for quantifying the results of immunoassays. For
example, U.S. Pat. No. 5,018,209 issued May 21, 1991 and U.S. Pat.
No. 5,008,185 issued Apr. 16, 1991, both to Bacus, describe digital
image processing methods and apparatus to analyze various features
of cells being viewed on a slide under a microscope. Because the
cells (or portions thereof) are randomly located on the slide, the
technician and system work in an interactive fashion whereby the
technician manually locates the cells on the slide that the system
thereafter analyzes. Thus, while the efficiency of the testing
process is increased by such an interactive system, it is not as
efficient as one which would automatically locate the region of
interest without human interaction.
U.S. Pat. No. 4,922,915 issued May 8, 1990 to Arnold, describes an
automatic image location method in the field of medical imaging
technology, such as computer tomography (CT) and magnetic resonance
imaging (MRI). In a typical diagnostic scan of a patient, several
reference samples of known optical density are placed in proximity
with the patient's body and are scanned simultaneously. These
images of the reference samples of known density are compared with
the images of various regions of the patient's body to determine
the relevant characteristics of those regions.
The method in Arnold is concerned with locating two regions: the
reference samples and the regions of interest within the patient's
body. With respect to the reference samples, the system locates the
samples automatically by two separate algorithms. The first
algorithm uses the fact that the reference samples are of known
optical densities. The Arnold system searches the entire digital
image for regions with these optical densities.
The second location algorithm uses pre-positioned metallic rods
proximately placed to the reference samples. Initially, the system
starts scanning the entire digital image for pixels of greatest
density. These pixels correspond to the metallic rods. Once the
rods are located, the reference samples are easily located because
the orientation of the samples in relation to the metallic rods is
predefined.
With respect to locating regions of interest in the patient's body,
the search performed by the Arnold system is not fully automated.
After the reference samples are located, Arnold requires that a
human operator define an enlarged region of interest, for example
around a bone structure, which the system thereafter refines. This
step in Arnold is necessary because the system is unable to exclude
regions which add error to the density readings.
While Arnold's method of automatically locating digital images
works well when regions are either of known densities or known
orientations, it is not satisfactory when the region of interest
has neither known intensity or position. In Arnold's method, human
interaction during the analysis step is always required.
The above-mentioned methods of digital analysis do not preform
quantitative analyses of specimens, but rather only locate a region
of interest based on optical density. However, in the analysis of
chemical and biological specimens, it is often the density of a
particular color that is the relevant measurement parameter. For
instance, some immunoassays employ labeled reagents, such as
certain colloidal reagents, that generate a color when an analyte
is detected in a biological specimen.
Therefore, it is an object of the present invention to provide a
system and method for automatic image location and quantitative
analysis when the region of interest is of neither known intensity
or position in the image.
Another object of the present invention is to provide a reliable
automated method for quantifying the results of an immunoassay
wherein the method provides a more accurate measurement that better
corresponds to the true concentration of an analyte in a fluid
sample compared with prior art methods that use densitometers and
reflectometers.
SUMMARY OF THE INVENTION
In summary, the present invention is a method and system for the
automatic analysis of a testing substrate for an analyte derived
from a specimen, such as a specimen of biological origin, when
neither the position nor the optical density (or color density) of
the region of the testing substrate where the analyte, if present,
is precisely known. The invention also provides a method for more
accurately measuring the concentration of the analyte in the
specimen.
The method comprises the steps of applying a liquid sample
suspected of containing an analyte to a testing substrate having an
immobilized thereon, in a limited region of the testing substrate,
a receptor capable of directly or indirectly binding to the
analyte. A labeled reagent, capable of binding directly or
indirectly to the analyte and generating a color signal, is added
to the testing substrate. If the analyte is present, a color is
generated at the area of the testing substrate where the receptor
is immobilized.
The testing substrate is then illuminated and, using electronic
equipment, a digital image of the testing substrate is acquired. In
preferred embodiments, the testing substrate is illuminated using
reflected light. The illumination is pre-calibrated to correct for
lighting intensity variations. The digital image is automatically
scanned to locate an area of the testing substrate having the
highest color density and generating a first measurement of color
density that corresponds to pixels per unit area. To aid in the
scanning of the digital image, one embodiment of the invention
employs a positional marker on the substrate (such as a dark or
colored circle, line, spot, or any other shape) to generally
indicate where the immobilized receptor is located. The use of a
positional marker reduces both the amount of time require to locate
the receptor area and the degree of error in locating the most
optically dense portion of the testing substrate.
Next, an area peripheral to the area of highest color density is
located and a second measurement of color density is generated that
corresponds to pixels per unit area. Because this peripheral area
is not proximate to the receptor area where the analyte, if present
in the sample, specifically binds, this area represents the
background density of the substrate, which can be considered to be
the background "noise" in the measurement of the analyte. In a
preferred embodiment, the second measurement is generated that
corresponds to the pixels per unit area in an annular region that
circumscribes the receptor area. The presence or absence of the
analyte in the liquid sample is calculated by adjusting the first
measurement with said second measurement in accordance with a
predefined mathematical function.
In some cases, the result of the test will be interpreted as a
binary, positive/negative result according to whether the
measurement taken is above or below a given threshold. In other
cases, a continuous range of values may be generated from different
samples that correspond to the concentration of the analyte present
in each tested sample.
BRIEF DESCRIPTION OF THE DRAWINGS
Additional objects and features of the invention will be more
readily apparent from the following detailed description and
appended claims when taken in conjunction with the drawings, in
which:
FIG. 1 is a block diagram of a system for optically analyzing
biological and other analytes.
FIG. 2 shows a sample holder with multiple analytes deposited
thereon.
FIG. 3 is a conceptual diagram of the method used to locate the
optically densest portion of an analyte deposited on a
substrate.
FIG. 4 is a flow chart of the steps of the present invention.
FIG. 5 is a block diagram of an alternate embodiment of a system
for analyzing biological and other analytes.
FIG. 6 shows the linear titration curve obtained using the optical
analyzer.
FIG. 7 shows relationship between linearity of titration curve and
amount of receptor bound to testing substrate.
DESCRIPTION OF THE PREFERRED EMBODIMENT
The method aspect of the invention involves the analysis and
measurement of assay results for the determination of the presence
or absence of an analyte in a sample, preferably a sample of
biological origin. The type of assay that is performed prior to the
measurement step can be one of many assays known in the art as long
as the assay employs a testing substrate that has a limited area to
which the analyte is concentrated, and a peripheral area to which
the analyte does not specifically bind. In a preferred embodiment,
the assay is an immunoassay for the detection of an analyte in a
biological sample wherein the testing substrate is a
liquid-permeable membrane which has a receptor bound on a limited
area thereof. The receptor is capable of specifically binding to
the analyte. This type of immunoassay is well known by those
skilled in the art and is described in further detail in U.S. Pat.
No. 5,006,464 to Chu et al., incorporated herein by reference.
In general, the analyte can be any substance that may be present in
a liquid sample that is to be detected by the assay procedure. In
preferred embodiments, the liquid sample is of biological origin,
such as urine, plasma, serum, whole blood, and the like. The liquid
sample may also comprise a diluent to which the contents of a
throat swab, vaginal swab, bacterial culture, or other biological
specimen has been added. Thus, in a preferred embodiment, the
analyte may be a "biological analyte", that is any substance
present in a biological sample that is sought to be detected. For
example, the analyte may be an antibody to a particular virus, such
as the Human Immunodeficiency Virus (HIV), Rubella, Herpes Simplex
Virus I and II (HSV I and HSV II), etc.; a bacteria such as
Streptococcus pyogenes or Neisseria gonorrhea; a hormone such as
human chorionic gonadotropin (HCG) or luteinizing hormone; a
protein, or any other detectable substance that may be present in a
biological sample. Numerous other possible analytes are listed in
U.S. Pat. No. 5,006,464 to Chu et al. Additionally, the assay may
test for the presence of more than one analyte. Thus, the term
"analyte" generally refers to one or more a specific type of
substance that is present in a sample. For example, in the case of
an assay for the diagnosis of pregnancy, the analyte is HCG.
However, numerous HCG molecules would be present in the serum or
urine sample of the patient. The term "analytes" or "more than one
analyte" refers to more than one specific type of substance in a
sample. For example, an assay that detects the presence of HSV I
and HSV II is an assay that detects the presence of more than one
analyte (i.e two different analytes).
The liquid sample is applied to a testing substrate. Any testing
substrate can be used as long a receptor that is capable of binding
to the analyte can be immobilized onto the testing substrate. Thus,
the testing substrate can be a porous or a non-porous surface. In a
preferred embodiment, the testing substrate is a porous surface
such as a fiberglass or nitrocellulose membrane through which the
liquid sample can flow. Typically, the testing substrate is an
exposed surface of an immunoassay device such as the device
described in U.S. Pat. No. 5,006,464 to Chu et al. Other suitable
immunoassay devices that employ porous testing substrates are well
known in the art.
A limited region of the testing substrate has a receptor
immobilized thereon that is capable of directly or indirectly
binding to the analyte. This region, which may take any shape, but
which is typically a small circle or dot, is hereinafter termed the
"receptor area". If a second analyte is to be tested
simultaneously, then there is a second receptor area on the
substrate that is separate and distinct from the first receptor
area. By way of example, if the analyte is a human antibody to HIV,
the receptor may be an HIV antigen. The HIV antigen is immobilized
onto a portion of the testing substrate using known methods. If
antibody to HIV is present in the liquid sample, it will
specifically bind to the HIV antigen. The remainder of the testing
substrate will not specifically bind to the antibody. Thus, after
the liquid sample is applied to the testing substrate, the analyte,
if present in the sample, will be concentrated at the receptor
area.
A labeled reagent, capable of binding directly or indirectly to the
analyte and generating a color signal, is added to the testing
substrate. If more than one analyte is being tested simultaneously,
more than one labeled reagent is added to the testing substrate,
either simultaneously or sequentially. The term "capable of binding
directly", means that the labeled reagents can specifically bind to
the respective analytes. For example, if the analyte is a human
IgG, the labelled reagent may be labelled antibody to human IgG, or
labelled protein A, which binds to the FC fragment of IgG. The term
"binding indirectly" means that at least one intermediate reagent
is added to the testing substrate that is capable of binding to the
analyte, and the labeled reagent is capable of binding to the
intermediate reagent. For example, if the analyte is human IgG, an
intermediate reagent may be a rabbit antibody to human IgG. The
labelled reagent may be a goat antibody that specifically binds to
the rabbit antibody.
The presence of bound analyte is indicated by the presence of color
that is generated by the labeled reagent. The label may be a
colored substance itself, such as colloidal gold which, when
concentrated at the receptor area, generates a red color.
Alternatively, the label may be an enzyme which, when substrate for
the enzyme is added, causes a colored product to be generated. A
variety of labels are well known in the art. Techniques are
well-known in the art for attaching labels to reagents. A preferred
embodiment of the invention employs a reagent labelled which
colloidal gold because an enzyme-substrate reaction is not required
to generate color, thus requiring fewer steps in performing the
assay, and high sensitivity can be achieved using colloidal
labels.
In order to measure the results of the assay, the testing substrate
is illuminated and, using electronic equipment, a digital image of
the testing substrate is acquired. In preferred embodiments, the
testing substrate is illuminated using reflected light. The
illumination is pre-calibrated to correct for lighting intensity
variations. The digital image is automatically scanned to locate an
area of the testing substrate having the highest color density,
i.e. the highest number of pixels per unit area, and generating a
first measurement of color density. To aid in the scanning of the
digital image, one embodiment of the invention employs a positional
marker on the substrate (such as a dark or colored circle, line,
spot, or any other shape) to generally indicate where the
immobilized receptor is located. Once the general region for the
immobilized receptor is identified, the region will be identified
for the area having highest color density. The use of a positional
marker reduces both the amount of time require to locate the
receptor area and the degree of error in locating the most
optically dense portion of the testing substrate.
Next, an area peripheral to the area of highest color density is
located and a second measurement of color density is generated as
determined by the number of pixels per unit area. Because this
peripheral area does not include any portion of the receptor area
(where the analyte, if present in the sample, specifically binds)
its measurement represents the background density of the substrate
and thus the "noise" in the measurement of the analyte. Background
color may result from non-specific binding of the analyte or
labeled reagent to areas of the testing substrate where there is no
receptor bound. If the assay is done properly, the background color
should be much less than the color generated at the receptor area
when analyte is present in the sample. However, in some cases, even
when assay procedures are done properly, portions of the peripheral
area can be as dark as the area where analyte has bound
specifically to receptor. This can sometimes happen if the
biological sample being tested has particulate matter that is
trapped by the membrane and unable to flow through. Samples that
have been frozen and thawed often contain particulate matter that
can lead to background problems.
High levels of background can also occur if the substrate is not
even and, as a result, aggregates in the sample pool at a
particular region of the substrate. In prior art immunoassay
methods that employ refractometers, a positive sample that produces
high background can be falsely diagnosed as negative if the area of
high background on the testing substrate coincides with the point
where background reflectance is measured. Because the present
invention uses digital technology to determine the number of pixels
per unit area, a larger area of background can be measured, not
just one point as with reflectance, and thus a more accurate
reading can be obtained than is possible with prior art
technologies. In a preferred embodiment the background density is
determined by measuring an annular region that surrounds the
receptor area is measured. If there is more than one receptor area,
two annular regions, one for each receptor area, would be measured.
Alternatively, a single large background region could be defined
for all receptor areas. The preferred approach for background
determination may vary depending upon the size of the receptor
areas, the type of sample to be tested and the nature of the flow
of the sample.
After the region of highest color density (first measurement) and
background density (second measurement) are determined, the
presence or absence of the analyte in the liquid sample is
calculated by adjusting the first measurement with the second
measurement in accordance with a predefined mathematical function
as exemplified in more detail below. If a positional marker is used
that is located within the peripheral area, then the second
measurement is adjusted so that the color intensity of the
positional marker is not included as part of the background
measurement. Because color intensity can sometimes vary depending
upon whether the testing substrate is wet or dry, the first and
second measurements can also be adjusted to take into consideration
this factor. For example, calibration spots on the testing
substrate could be used that have a fixed color intensity,
independent of the sample being analyzed, but dependent on whether
the testing substrate is wet or dry. The calibration spot could
also serve a dual purpose as a positional marker.
The result of the test can be interpreted as a binary,
positive/negative result according to whether the measurement taken
is above or below a given threshold. In preferred embodiments, the
adjusted measurement, which corresponds to an adjusted number of
pixels per unit area, will be quantitative, correlating to the
concentration of analyte present in the sample. Typically, for
quantitative measurements, a standard calibration curve of a known
sample would be generated and used to determine the concentration
of the sample tested based on the measurements generated from the
test sample.
In describing the system aspect of the present invention, reference
is made to FIG. 1, where there is shown a block diagram of the
system for optically analyzing biological and other analytes
designed in accordance with the principles of the present
invention. System 100 includes a central processing unit (CPU) 102,
computer memory 104, user interface 106, system communication bus
108, and an optical measurement subsystem 110. The optical
measurement subsystem 110 includes a shadowless (i.e., uniform)
light source 112, camera 114, and camera interface 116. The optical
measurement subsystem 110 is typically enclosed in a housing 118 so
that optical images of analytes can be obtained under controlled
optical conditions.
Memory 104, which will typically include both random access memory
and secondary memory such as magnetic disk storage mechanisms, is
sufficiently large enough to store a plurality of digital images
120, analyte measurement program 122, and a light source
calibration program 124. Alternatively, programs 122 and 124 could
be stored on Read Only Memory (ROM) chips.
It should be noted that the specific memory devices used are not
important to the operation of the present invention so long as they
have sufficient capacity and operating speed to enable the optical
image analysis tasks described below.
The user interface 106 will typically include at least one output
communication device such as a printer 125 and/or monitor 126 for
communicating the results of tests conducted by the system, and at
least one input communication device such as keyboard 127 and/or
mouse pointer device 128. Many other combinations of user
interfaces could be used, and the specific interfaces shown in FIG.
1 should not be construed as a limitation.
Before any testing substrates are analyzed, light source 112 is
calibrated. Calibration is performed each time the system is
powered on, and may need to be performed periodically if the system
is kept on for long periods of time, because any changes in the
light source's intensity could affect the result of the test. For
example, if the measurement produced by the test is greater than a
certain threshold value, the result of the test may be deemed
positive. Otherwise, the test may be negative. These threshold
values, stored as constants (or as a mathematical formula) in the
analyte measurement program 112, are based upon a certain light
intensity level. Without proper calibration, the possibility of
false test results increases.
In the preferred embodiment, calibration is accomplished by
calibration program 124 by measuring the light source's intensity
with a light sensor (see light sensor 186 in FIG. 5). In an
alternate embodiment of the invention, the calibration program 124
takes as input an image of a calibration substrate. In the
preferred embodiment a calibration substrate is a regular testing
substrate without any analyte deposited on its surface. The
intensity of light is measured according to the average density of
the image of the calibration substrate. Then, a calibration
coefficient is computed by dividing the average image density with
a predefined standard value. All subsequent image density values
are multiplied by this calibration coefficient.
When system 100 is ready for operation, camera 114 takes an analog
image of at least a portion of the top surface of test carrier 129.
The top surface of carrier 129, as depicted in FIG. 1, includes
testing substrate 130 on which is deposited a chemically active
reagent. Prior to insertion of carrier into the optical measurement
subsystem 110, an analyte 132, typically derived from a biological
specimen, is deposited onto the region of the test carrier where
substrate 130 is located. In the preferred embodiment, the chemical
interaction of the analytes 132 with the chemically active
substrate 130 typically causes the analyte 132 to be the optically
densest portion of the substrate 130.
FIG. 2 shows a test carrier 129' with multiple analytes 144
deposited on its substrate 130. Positional marker 146 gives the
relative position of substrate 130 on carrier 129'. As previously
mentioned, marker 146 may be any shape or size sufficient to
indicate the general position or location of the substrate 130
and/or the analyte(s) on the test carrier 129'.
The analog image generated by camera 114 is digitized by camera
interface 116 and sent via bus 108 to memory 104 where the digital
image is stored as an array of pixel values. In the presently
preferred embodiment, the portion of the test carrier 129 captured
by the camera 114 is 5/16".times.5/16" and is represented as a
170.times.170 array of pixel elements. Additionally, the presently
preferred embodiment has the capability to process both color and
gray scale images, with 8-bit pixels (256 gray scale levels) being
used for gray scale images and 24-bit pixels (256.sup.3 levels)
being used for color images. It should be appreciated that the
image could be formed from more or less pixel elements and more or
less scale image levels to alter the image resolution and
sensitivity. It will also be appreciated that other data structures
for image storage are possible.
Note that for some analyte measurement tests the measured "density"
of the analyte and background regions of the digital image will be
the total optical density of a portion of the digital image, while
for other analyte measurement tests the measured density will be
the density of a particular color. That is, when the digital image
is a color image each pixel will be represented by Red, Green and
Blue (RGB) values, and the test measurements can be based on any
one or predefined combination of the three RGB color values for the
image's pixels. Thus, the term "density" in the discussions below
concerns the density of a preselected optical characteristic of the
digital image which is relevant to the measurement being
performed.
After the digital image has been captured, the image is analyzed by
analyte measurement program 122. This analysis includes locating
the analyte by locating within the digitized image a circular
region of predefined size having the greatest optical density,
locating a "background" region of the substrate 130 that is not
covered by the analyte, and computing a test result by computing a
predefined mathematical function of the average density of the
background region and the average density of analyte region. The
mathematical function may be as simple as subtracting the
background density from the analyte density, or may be a
considerable more complex function. In the preferred embodiment,
the circular region of greatest optical density is sized to be
small enough so as to be entirely covered the smallest anticipated
analyte, and thus the average optical density of the circular
region should be representative of the optical density of the
chemically reacted analyte.
To accomplish this analysis, analyte measurement program 122
performs three main processes: pre-scan, fine-scan, and background
density compensation. It will be appreciated that program 122
executes differently according to whether a positional marker is
included on the test carrier 129 or not. Program 122 takes the
digital image stored in memory 104 as input and begins a raster
scan of the entire image. If a positional marker is present,
program 122 then performs a raster scan across the image to locate
the marker, which will typically be either the pixels of greatest
density, or pixels of a particular color. From the orientation of
the marker, program 122 will determine a smaller region of
interest. Thereafter, program 122 confines its pre-scan and
fine-scan processes to this defined region. If no positional marker
is present on the substrate, then program 122 executes its pre-scan
and fine-scan processes on a predefined region of the digital
image.
The pre-scan process searches the region of interest for an area of
greatest average density. This area will correspond to the reaction
of the analyte to the reagent. The area should be as large as
possible while still fitting entirely within the site of the
analyte and should be sufficiently small to avoid noise
sensitivity. In the preferred embodiment, the portion of the
digital image used to determine the optical density of the analyte
is a circle of diameter 64 pixels across.
FIG. 3 is a conceptual diagram of the pre-scan process used to
approximately locate the densest area of the analyte 132 deposited
on the substrate 130. Please note that FIG. 3 is not drawn to
scale, and that the analyte 132 will typically cover a much smaller
fraction of the substrate 130 than shown in this conceptual
representation of the scanning process. The pre-scan process
measures and compares the densities of a sequence of circular
regions 160 arranged in columns and rows, where the centers of the
columns are spaced apart by a distance of .DELTA.X and the centers
of the rows are spaced apart by a distance of .DELTA.Y. By
comparing the densities of these regions the prescan process
determines center of the circular region 160 of greatest density,
as represented by circular region 162, and thereby locates the
approximate center of the analyte. Table 1 contains a pseudocode
representation of this process.
TABLE 1 ______________________________________ PRESCAN PROCESS
______________________________________ Specify range of regions to
be tested: X1, Y1 = center of top-left region to be tested X2, Y2 =
center of bottom-right region to be tested CX, CY and CD represent
the position and density of the region of greatest optical density
found so far. CX = X1 CY = Y1 CD = 0 For X = X1 to X2, by steps of
size .DELTA.X { For Y = Y1 to X2, by steps of size .DELTA.Y {
Measure density D of region centered at X,Y within a radius of Z
pixels Update center value whenever higher density region is found
If D > CD { CX = X CY = Y CD = D } }
______________________________________
After the region of greatest optical density is approximately
found, the fine-scan process is used to more precisely locate the
reaction region of greatest optical density. The fine-scan process
takes as its input the center of the area of greatest density
obtained from the prescan process. The center of the area is then
shifted by one or two pixel elements in both the X and Y
coordinates. The densities of these resulting areas are then
calculated by summing the pixel element readings for the areas. The
measured optical or color density for each area is compared with
the greatest density located by the pre-scan process, and the
greatest density area is accordingly updated. The fine-scan process
then computes the average pixel density for the area by taking the
greatest density reading and dividing by the number of pixels in
the circle. This average density is the output result of the
fine-scan procedure. Table 2 contains a pseudocode representation
of this process.
TABLE 2 ______________________________________ FINE-SCAN PROCESS
______________________________________ Initialize the starting X
and Y ranges from the center of the region found in Prescan.
X.sub.-- START = CX Y.sub.-- START = CY Let S1, S2, S3, S4, S5 and
S6 be relatively small integer values greater than zero. For X =
X.sub.-- START - S1 to X.sub.-- START + S2, by steps of S3 { For Y
= Y.sub.-- START - S4 TO Y.sub.-- START + S5, by steps of S6 {
Measure density D of region centered at X,Y within a radius of Z
pixels. Update center value whenever higher density region is
found. If D > CD { CX = X CY = Y CD = D } }
______________________________________
After the fine-scan process refines the center coordinates of the
region of greatest density, the analyte measurement program 122
then performs a background compensation step. To obtain the
necessary background reading, annular region 136, as shown in FIG.
1, is selected by program 122. Annular region 136 has an inner
radius sufficiently large such that none of the analyte 132 is
found in region 136. An average pixel density for annular region
136 is computed as discussed above. The measured density of the
analyte is then adjusted in accordance with the measurement
background region density. In some cases the adjustment is simply a
subtraction operation, while in others it may be accomplished by
division or other mathematical operation.
This adjusted measurement value may be interpreted as the result of
the test according to the nature of the reaction of the analyte
with the reagent. In some cases, a simple threshold test will
result. That is, if the adjusted measurement value is greater than
a pre-determined threshold, then the test is considered positive.
Otherwise, the test is considered negative. Alternatively, the
adjusted measurement value may be a value in a continuous range of
values that is to be interpreted by the user, or that is mapped
with respect to a predefined scale and then presented for
interpretation by the user. Thus, the adjusted measurement value
may correspond to concentration of analyte present in the
sample.
The flow chart in FIG. 4 represents the sequence of steps used to
test an analyte, from depositing the analyte onto the substrate
through generation of the final measurement value.
An alternate embodiment of the present invention is depicted in
FIG. 5. Instead of a positional marker being affixed to individual
carriers, as shown as marker 146 in FIG. 2, a separate template 180
is positioned between camera 114 and carrier 129. The image of
template 180 is thus superimposed upon the image of carrier 129
when the image is captured. To insure the proper alignment of the
two images, template 180 would remain in a fixed location; while
carrier 129 would be slid into position by way of guide rails 182.
The image of template 180 would be used to mark the region where
the analyte is deposited on the substrate. Use of the template
would obviate the need to place position markers on individual
carriers.
Also depicted in FIG. 5, a light sensor 186 can be positioned
inside the measurement subsystem's housing 118 to measure the
intensity of light emitted from light source 112. In this
embodiment, readings from sensor 186 are used to calibrate the
light source. This makes the calibration process totally automatic
and thus the user is not required to perform or assist with the
calibration process.
It will be appreciated that, although the presently preferred
embodiment is currently used for the detection of antibodies to the
human immunodeficiency virus (HIV), that application area is one of
many potential applications and should not be construed as a
limitation. In fact, the method and system of the present invention
is broad enough to include the automatic testing of any analyte
that visually reacts with any reagent.
It will further be appreciated that the present invention overcomes
problems in prior automated systems. Specifically, the present
invention does not require a human operator to work interactively
at the analysis phase with the system to indicate the regions of
interest for testing. Likewise, the present invention is able to
locate the specific regions of interest without precisely knowing
in advance either their location or their optical densities on the
digital image.
EXAMPLE 1
Generation of Standard Curve
The exposed membrane of an immunoassay testing device was
pretreated with a 1:10 dilution of normal human serum in phosphate
buffered saline and dried. Protein A-colloidal gold at
concentrations of 14.2 ng/.mu.l, 7.12 ng/.mu.l, 3.56 ng/.mu.l and
1.78 ng/.mu.l was inoculated onto the pretreated membranes in
triplicate (i.e. 3 separate devices for each concentration). The
digital readings generated by the colloidal gold was measured using
the optical analyzer system described herein. The results were
tabulated and plotted and are shown in FIG. 6. The results
demonstrate that the reading obtained from the optical analyzer is
concentration dependent. Thus, a measurement obtained from a sample
having an unknown concentration of analyte can be compared to this
type of standard curve to generate quantitative results.
EXAMPLE 2
Effect of Concentration of Antigen on Testing Substrate
Recombinant rabbit-anti-HIV recombinant protein antiserum
(Rb.alpha.HIV), having a concentration of approximately 125
.mu.g/ml, was diluted with normal human serum using 2.times. serial
dilutions. The diluted Rb.alpha.HIV samples were added to the
membranes of immunoassay devices having either 0.5 .mu.l or 1.0
.mu.l HIV recombinant antigen inoculated at the receptor area. A
Protein A-colloidal gold conjugate was added to each immunoassay
device. Measurements were obtained for each dilution of
Rb.alpha.HIV using the optical analyzer described herein. The
results were tabulated and plotted and are shown in FIG. 7. The
results demonstrate that at low concentration of analyte
(Rb.alpha.HIV) there is not much difference between the low (0.5
.mu.l/membrane) and high (1.0 .mu.l/membrane) concentrations of
antigen at the receptor area. However, at higher concentrations of
analyte, sensitivity increases with increased concentration of
antigen at the receptor area. Standard curves like this can be
prepared so that values from test samples can be compared with
values of samples having known concentrations to generate
quantitative information about the test sample. Standard curves can
also be generated from known samples that have been assayed and
allowed to dry onto the testing substrate prior to measurement,
thus correcting for variation in measurements that can occur
between wet and dry testing substrates.
While the present invention has been described with reference to a
few specific embodiments, the description is illustrative of the
invention and is not to be construed as limiting the invention.
Various modifications may occur to those skilled in the art without
departing from the true spirit and scope of the invention as
defined by the appended claims.
* * * * *